Macrophage-Neutrophil Co-Culture Assay

The herein described macrophage-neutrophil co-culture assay was adapted from Marwick et al. [24 (link)]. Macrophage differentiation was induced by cultivation of isolated monocytes in RPMI 1640 medium supplemented with GlutaMAX™, 10% FCS and 100 ng/ml M-CSF (130-096-491, Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 days, with medium change performed on the third day. One day before the co-culture experiment, monocyte-derived macrophages (MDMs) were harvested and re-seeded in 48-well-plates at 1 × 10⁵ cells per well. To induce a pro-inflammatory phenotype, MDMs were stimulated with 20 ng/ml interferon-gamma (INFy) (Imukin®) (Boehringer Ingelheim, Vienna, Austria). On the day of co-culture, MDMs were washed twice with 1 × DPBS, before autologous neutrophils were added at a 1:5 or 1:10 ratio (MDMs:neutrophils) in RPMI 1640 medium, without FCS and M-CSF. In indicated experiments, neutrophils were cultured for 24 h before co-culture assays in order to obtain apoptotic neutrophils. After 30 min of co-culture at 37 °C, cells were stimulated with 1 ng/ml lipopolysaccharide (LPS) (L4391, Sigma-Aldrich) and cultivation was continued for 6 or 18 h. Supernatants were harvested after 6 h by centrifugation at 450 × g and 7000 × g and frozen at −80 °C. MDM phenotype was assessed after 18 h of co-culture by flow cytometry.